Cerebral cavernous malformations (CCMs) are vascular lesions of the central nervous system that cause hemorrhagic stroke, focal neurological deficits and seizures. CCM lesions appear as grossly dilated capillaries that grow into late-stage, multicavernous structures devoid of intervening parenchyma. Tight junctions between the endothelial cells lining the CCM caverns are defective, increasing the permeability of the blood- brain barrier. CCM occurs as both familial and sporadic cases. Germline mutations in any one of three genes causes the familial form of the disease: CCM1 (KRIT1), CCM2 (malcavernin), and CCM3 (PDCD10). In vitro knockdown of any one of the CCM genes activates the RhoA pathway, specifically through Rho Kinase (ROCK). Epidemiology of CCM and somatic mutation analysis indicate the disease follows a two-hit mutation mechanism where two inactivating mutations in the same gene are required for lesion genesis. Only late-stage CCM lesions are resected from patients once they start causing clinical symptoms, so mouse models of the disease were developed using the two-hit mutation hypothesis to study lesions at earlier stages of development. These mice develop late-stage, multicavernous CCM lesions as well as early-stage isolated caverns, making this the first model capable of tracking CCM lesions from genesis through the early-stages and into the late-stages. I propose to determine the molecular and genetic mechanisms causing CCM lesion genesis and progression. Specific Aim 1) To study the effects of ROCK inhibition on mouse CCM lesion progression. I hypothesize that ROCK activation is an upstream even in the progression of lesions from the early-stage isolated caverns to late-stage multicavernous structures. I will treat our CCM mice with the specific ROCK inhibitor fasudil to determine if inhibiting ROCK can halt lesion growth. I will evaluate mice for the total number and size of lesions. Additionally, I will examine the lesions from fasudil-treated and control mice for alterations in the downstream histopathological phenotypes associated with the late-stage lesion. If ROCK activation is a key step in CCM pathogenesis, then fasudil treatment will abrogate these downstream phenotypes and halt lesion growth at an earlier stage. Specific Aim 2) To investigate the two-hit mechanism in early-stage mouse CCM lesions. While there is evidence for somatic mutations in late-stage human lesions, it is still unclear if somatic mutations cause lesion genesis. I propose to examine CCM lesions using immunohistochemistry for the somatic loss of CCM protein expression in early- and late-stage lesions. Furthermore, I will examine these lesions directly for somatic mutations using deep resequencing. The presence of somatic mutations and mosaic somatic loss of CCM protein staining within the early-stage lesions will indicate that CCM lesion genesis is caused by a two-hit mutation mechanism. Together these aims will elucidate the molecular and genetic mechanisms causing CCM lesion genesis and progression.